Localization of the ligand binding site of the neurokinin-1 receptor: interpretation of chimeric mutations and single-residue substitutions

RR Huang, H Yu, CD Strader and TM Fong

Department of Molecular Pharmacology and Biochemistry, Merck Research Laboratories, Rahway, New Jersey 07065.

Previous studies have indicated that substitution of the third or fourth extracellular segment of the human neurokinin-1 receptor with the equivalent segment from the neurokinin-3 receptor affects the binding affinities of peptide agonists and/or nonpeptide antagonists. To elucidate the roles of the divergent residues within these domains in ligand binding, single-residue substitutions in these regions were analyzed. Neurokinin B affinity was increased by one single-residue substitution (E172K) in the third extracellular segment and one double- residue replacement (L279R+K280W) in the fourth extracellular segment, and the affinity for the antagonist L-703,606 was reduced by one single- residue substitution (Y272A) in the fourth extracellular segment. The effect of these three specific substitutions is consistent with the prediction of chimeric mutations. However, the substantial reduction in ligand binding affinity observed upon multiple-residue substitutions in the third extracellular segment (residues 176-183 or 187-195) has not been reproduced by eliminating potential electrostatic interactions or substituting with the corresponding residues from the neurokinin-3 receptor, suggesting that the reduction in ligand binding affinity observed with some chimeric receptors is not due to the loss of direct electrostatic interactions. These data indicate that other factors such as conformational effects may complicate the interpretation of data obtained with chimeric receptors, and they demonstrate the need to evaluate chimeric receptors along with single-residue substitutions in the same region to localize specific residues involved in ligand binding. Furthermore, the available data suggest that one major determinant of peptide selectivity in the neurokinin-1 receptor may be the conformational compatibility between a peptide and the receptor.

Volume 45, Issue 4, pp. 690-695, 04/01/1994
Copyright © 1994 by American Society for Pharmacology and Experimental Therapeutics

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